Evaluation of Four Strategies for SARS-CoV-2 Detection: Characteristics and Prospects

ABSTRACT The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed an enormous burden on the global public health system and has had disastrous socioeconomic consequences. Currently, single sampling tests, 20-in-1 pooling tests, nucleic acid point-of-care tests (POCTs), and rapid antigen tests are implemented in different scenarios to detect SARS-CoV-2, but a comprehensive evaluation of them is scarce and remains to be explored. In this study, 3 SARS-CoV-2 inactivated cell culture supernatants were used to evaluate the analytical performance of these strategies. Additionally, 5 recombinant SARS-CoV-2 nucleocapsid (N) proteins were also used for rapid antigen tests. For the wild-type (WT), Delta, and Omicron strains, the lowest inactivated virus concentrations to achieve 100% detection rates of single sampling tests ranged between 1.28 × 102 to 1.02 × 103, 1.28 × 102 to 4.10 × 103, and 1.28 × 102 to 2.05 × 103 copies/mL. The 20-in-1 pooling tests ranged between 1.30 × 102 to 1.04 × 103, 5.19 × 102 to 2.07 × 103, and 2.59 × 102 to 1.04 × 103 copies/mL. The nucleic acid POCTs were all 1.42 × 103 copies/mL. The rapid antigen tests ranged between 2.84 × 105 to 7.14 × 106, 8.68 × 104 to 7.14 × 106, and 1.12 × 105 to 3.57 × 106 copies/mL. For the WT, Delta AY.2, Delta AY.1/AY.3, Omicron BA.1, and Omicron BA.2 recombinant N proteins, the lowest concentrations to achieve 100% detection rates of rapid antigen tests ranged between 3.47 to 142.86, 1.74 to 142.86, 3.47 to 142.86, 3.47 to 142.86, and 5.68-142.86 ng/mL, respectively. This study provided helpful insights into the scientific deployment of tests and recommended the full-scale consideration of the testing purpose, resource availability, cost performance, result rapidity, and accuracy to facilitate a profound pathway toward the long-term surveillance of coronavirus disease 2019 (COVID-19). IMPORTANCE In the study, we reported an evaluation of 4 detection strategies implemented in different scenarios for SARS-CoV-2 detection: single sampling tests, 20-in-1 pooling tests, nucleic acid point-of-care tests, and rapid antigen tests. 3 SARS-CoV-2-inactivated SARS-CoV-2 cell culture supernatants and 5 recombinant SARS-CoV-2 nucleocapsid proteins were used for evaluation. In this analysis, we found that for the WT, Delta, and Omicron supernatants, the lowest concentrations to achieve 100% detection rates of single sampling tests ranged between 1.28 × 102 to 1.02 × 103, 1.28 × 102 to 4.10 × 103, and 1.28 × 102 to 2.05 × 103 copies/mL. The 20-in-1 pooling tests ranged between 1.30 × 102 to 1.04 × 103, 5.19 × 102 to 2.07 × 103, and 2.59 × 102 to 1.04 × 103 copies/mL. The nucleic acid POCTs were all 1.42 × 103 copies/mL. The rapid antigen tests ranged between 2.84 × 105 to 7.14 × 106, 8.68 × 104 to 7.14 × 106, and 1.12 × 105 to 3.57 × 106 copies/mL. For the WT, Delta AY.2, Delta AY.1/AY.3, Omicron BA.1, and Omicron BA.2 recombinant N proteins, the lowest concentrations to achieve 100% detection rates of rapid antigen tests ranged between 3.47 to 142.86, 1.74 to 142.86, 3.47 to 142.86, 3.47 to 142.86, and 5.68 to 142.86 ng/mL, respectively.

However, the methods used and results presented should be elaborated. I have several comments/queries to the current version of the manuscript. The authors should respond them one by one before the manuscript is accepted for publication.
Major comments -line 142: need to describe the purposes of the two systems used, (1) RT-qPCR, (2) ddPCR. (1) for reference NAAT test? (2) for quantifying the viral load in copies/mL? If (1) was used as the reference, the results should also be presented. -line 149: need to mention explicitly here, 'The recombinant N proteins were measured ........ '. -line 158: to be clear, insert the term 'serial two-fold dilution' -line 161: need to elaborate the two dilution factors, 1:61 and 1:241 as 50uL in 3050uL and 50uL in 12050uL respectively -lines 167-169: add numbers 1-5 before describing the name of each assay - Table 1: need to add the two sub-headings (1) real-time RT-PCR and (2) POCT -lines 172-178: need to briefly describe the principles and hands-on procedures, not everyone use this POCT. as you quote ID NOW as an example (ref 32) in line 301, you may use this technique to introduce your POCT.
-lines 179-191: add numbers 1-19 before describing the name of each assay, need to briefly describe the principles of the rapid antigen tests. (1) all were based on lateral flow principles, (2) antigen-antibody immune-complexes were based on fluorescence, latex, colloidal gold, (3) need instrument to read results or interpreted by naked eye - Table 1: need to assign number 1-19 for all rapid antigen tests, the number used should be concordant to those mentioned in the text, lines 179-191 and Table 2. -line 199: 'two laboratories', typo error? should be laboratory technicians?, you mentioned that your study was a single-centre evaluation (line 133) -lines 201-203: the author have to use examples to illustrate how to calculate the values of (1) lowest concentration, (2) concentration equivalent to 50uL, I suggest one example (e.g. Daan) from NAAT and one example for rapid antigen test (e.g. test 1), if there is limited of words required by the journal, these illustrations maybe put into supplementary section.
-the authors have to provide the raw data in supplementary section: (1) Rt-pcr no pooling, list out the test results for the 9 dilution points (2) Rt-pcr with pooling, list out the test results for the 9 dilution points (3) POCT, list out the test results for the 9 dilution points (4) rapid antigen tests, supernatants, list out the test results for the 9 dilution points (5) rapid antigen tests, recombinant N proteins, list out the test results for the 8 dilution points - Table 1, need to explain the ways in measuring the volume of extraction solution, mentioned by the kit insert? As far as I know, the extraction solution used by the rapid antigen kits were not mentioned from the kit insert explicitly. obtained from suppliers? measured by pipettes? -figure 2: I have no idea how can you come up this figure, you either elaborate the methods in the main text or delete it -discussion section: need to discuss the sensitivity difference between RT-PCR, POCT and rapid antigen tests for the results obtained in this evaluation Minor comments -line 68: need to mention the time for the first detection of the two variants in your country.
-the authors forgot to cite table 4 in the main text Reviewer #2 (Comments for the Author): The study by Chen and co-workers is well conducted and relevant as a reference for SARS-CoV-2 diagnosis by a more efficient strategy, especially since it demonstrates the efficacy of several RT-qPCR and antigen tests for Omicron detection. The introduction section is lengthy, but results description is very limited, the variations in detection from different tests is not explored (this is especially relevant for the antigen tests in which a great variation can be observed in tables) and the figure 2 besides being hard to follow, the way it is presented makes it difficult to have a comprehensive appreciation of the results. Authors should say how the limit of detection for each test was calculated.
Minor issues: 1) Table 4 is not mentioned withing the text 2) The sentence: "Mutations can occur continuously during virus transmission..."(lane 63) should be rephrased to: "naturally occurring mutations can be selected continuously during virus transmission..." Staff Comments:

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The study by Chen and co-workers is well conducted and relevant as a reference for SARS-CoV-2 diagnosis by a more efficient strategy, especially since it demonstrates the efficacy of several RT-qPCR and antigen tests for Omicron detection. The introduction section is lengthy, but results description is very limited, the variations in detection from different tests is not explored (this is especially relevant for the antigen tests in which a great variation can be observed in tables) and the figure 2 besides being hard to follow, the way it is presented makes it difficult to have a comprehensive appreciation of the results. Authors should say how the limit of detection for each test was calculated.
Answer 4: Thank you for your valuable suggestion.
We have revised the sentence to "Remarkably, this step with positive supernatants diluted in sample preservation solution introduces a pre-dilution effect (1:61 as 50 μL in 3050 μL for the single sampling test and 1:241 as 50 μL in 12050 μL for the 20-in-1 pooling test), leading to a loss of sensitivity as claimed by the reagent manufacturers." in the "Marked Up Manuscript -For Review Only" (Introduction, Lines 132-135).
Answer 5: Thank you for your valuable suggestion.
For ease of communication throughout the article, each real-time RT-PCR kit was assigned a code from Kit N01 to Kit N05, POCT was encoded as Kit P01, and each rapid antigen test was assigned a code from Kit A01 to Kit A19. "N", "P" and "A" are the initials of nucleic acid, POCT and antigen, respectively. We have also replaced the kit names in tables and in the Results and Discussion sections with their respective codes.  (2) POCT.
Answer 6: Thank you for your valuable suggestion.
We have added two sub-headings "Real-time RT-PCR tests" and "Point-of-care test" in the "Marked Up Manuscript -For Review Only" ( Answer 9: Thank you for your valuable suggestion. We have added a column named "Code" to Table 1 in order to code each detection kit in the "Marked Up Manuscript -For Review Only". The codes were concordant with those mentioned in the main text and were used for the following Results and Discussion sections. Therefore, we have added the footnote in For the single sampling tests and 20-in-1 pooling tests, 50 μL cell culture supernatants were added to 3 mL and 12 mL sample preservation solution, resulting in 61-fold and 241-fold dilutions of the initial sample concentration, respectively. The "concentration equivalent to the 50 μL sample" in Table 3 was the lowest concentration to achieve 100% detection rates of the 50 μL original sample before being added to the sample preservation solution. The "lowest concentration at 100% detection rates" was the lowest concentration to achieve 100% detection rates of the diluted sample after being added to the sample preservation solution, and was 1/61 (single sampling tests) or 1/241 (20-in-1 pooling tests) of the 50 μL original concentration.
For the point-of-care test, 50 μL cell culture supernatants were added to 500 μL sample extraction solution, resulting in an 11-fold dilution of the initial sample concentration. Similar to the rRT-PCR kits, the "lowest concentration at 100% detection rates" was 1/11 of the "concentration equivalent to the 50 μL sample" in Table 3.
For the rapid antigen tests, 50 μL cell culture supernatants or recombinant N proteins were added to different volumes of sample extraction solution for different antigen tests. Taking BGI as an example, 50 μL cell culture supernatants or recombinant N proteins were added to 400 μL sample extraction solution resulting in a 9-fold dilution of the initial sample concentration. Therefore, the "lowest concentration at 100% detection rates" was 1/9 of the "concentration equivalent to the 50 μL sample" in Table 3 and Table 4.
In order to describe the pre-dilution ratio and the calculation of the LOD for each kit more clearly, we have added " Table 2 Calculation of pre-dilution ratio of different detection methods."

Comment 12:
The authors have to provide the raw data in supplementary section: (1) Rt-pcr no pooling, list out the test results for the 9 dilution points (2) Rt-pcr with pooling, list out the test results for the 9 dilution points (3) POCT, list out the test results for the 9 dilution points (4) rapid antigen tests, supernatants, list out the test results for the 9 dilution points  Fifteen manufacturers specified the volume of extraction solution in their instructions.
For the remaining four antigen kits (Jinwofu, ACON, Orient Gene and Zybio) without mentioning the volume of extraction solution, we would consult the reagent manufacturers and verify by manual pipettes.
Because we added Table 2 to the article, and the volumes of extraction solution of different rapid antigen tests were moved to Table 2. Therefore, we added the footnote in  To reduce unnecessary confusion, we decided to use tables mainly to present the results and delete Figure 2. Table 3 and Table 4 showed the lowest concentrations of samples to achieve 100% detection rates. Besides, to provide more detailed information in the study, we have added Supplementary Material File 3 attached with the raw data of test results.
Comment 15: discussion section: need to discuss the sensitivity difference between

RT-PCR, POCT and rapid antigen tests for the results obtained in this evaluation
Answer 15: Thank you for your valuable suggestion.
We supplemented the discussion for the sensitivity difference between RT-PCR, POCT and rapid antigen tests. Correspondingly, the test results were analyzed in Results according to the comment of another reviewer. The revisions are as follows: (1) Real-time RT-PCR Results: At first, we compared the sensitivity difference between the single sampling tests and 20-in-1 pooling tests. As shown in the Results section, for single sampling tests and five rRT-PCR detection kits, the lowest inactivated virus concentrations to achieve 100% detection ranged between 1.28×10 2 -1.02×10 3 , 1.28×10 2 -4.10×10 3 , and Discussion: Therefore, we discussed the results in the "Marked Up Manuscript -For Review Only": When using five rRT-PCR kits to detect the same strain, the range of lowest inactivated virus concentrations to achieve 100% detection by 20-in-1 pooling tests was substantially the same as single sampling tests. Overall, when detecting the diluted samples higher than the claimed LODs, 20-in-1 pooling tests showed comparable analytical sensitivity to the single sampling tests (Discussion, Lines 298-302). Therefore, the 20-in-1 pooling tests enable the mass nucleic acid tests with only 5% of the original testing workload, greatly improving daily testing efficiency, which is the first consideration in the screening of asymptomatic infected cases in low-risk regions (Discussion, Lines 304-307).

(2) Point-of care test
Results: Unexpectedly, the Kit P01 showed comparable analytical sensitivity to rRT-PCR kits with the lowest concentration of 1.42×103 copies/mL for all three strains in the detection of SARS-CoV-2 in the study (Results, Lines 212-214).
Discussion: Therefore, we concluded in the "Marked Up Manuscript -For Review Only": With short turnaround time, portable procedures, and analytical sensitivity comparable to the gold standard rRT-PCR, nucleic acid POCT is a promising diagnostic agent for aiding testing expansion and can be used as confirmatory testing where laboratory-based nucleic acid amplification test is not available (Discussion, Lines 323-327).
Different from the rRT-PCR kits, great variations in analytical sensitivity were observed among rapid antigen kits. The lowest concentrations that achieved a 100% rate of detection success ranged from 10 4 to 10 6 copies/mL for inactivated cell culture supernatants and from 1 to 150 ng/mL for recombinant N proteins (Results, Lines 236-239).
Discussion: Therefore, we discussed the results in the "Marked Up Manuscript -For Review Only": Our study revealed that the detection performance of different rapid antigen kits varied greatly, and the lowest concentrations that achieved a 100% rate of detection success ranged from 10 4 to 10 6 copies/mL for inactivated cell culture supernatants and from 1 to 150 ng/mL for recombinant N proteins, which was essentially consistent with previously published articles. The antibody labeling method of rapid antigen tests would affect their analytical performance, and the fluorescence microsphere was observed to enhance the sensitivity of analytical signal by 10 to 100 folds compared to latex microsphere and colloidal gold (40). Besides, the lowest concentrations of the 50 μL original sample at 100% detection of rapid antigen tests (10 5 -10 7 copies/mL) were about 100 times as high as those of the single sampling test (10 3 -10 5 copies/mL) (Discussion, Lines 336-346   Because we added Table 2 to the article, Table 4 became Table 5 accordingly. We cited Table 5 in the "Marked Up Manuscript -For Review Only" (Discussion, Lines 382): In conclusion, the study evaluated the analytical sensitivity of four SARS-CoV-2 detection strategies applied in different settings and provided helpful insights into the scientific deployment of these tests (Table 5). beginning of outbreaks, the single sampling test of rRT-PCR was regarded as the most sensitive and specific method in detecting SARS-CoV-2, which was recommended for confirming infected cases and testing specific groups including contacts of confirmed or frequently exposed groups to ensure the timely implementation of public health measures and patient management procedures such as contact tracing and quarantine (Discussion,. However, the primary bottleneck of pooled testing is the reduced concentration of viral genetic material below the limit of detection (LOD) for a certain test due to sample dilution, thereby leading to decreased diagnostic sensitivity and false-negative results (Discussion, Lines 295-298).

Reviewer
Next, we further analyzed the test results and supplemented the Results section. Then, the corresponding conclusions were added to the Discussion section. (
Kit N03 reliably detected as low as 10 2 copies/mL for both sampling strategies, showing the most sensitive performance compared with other rRT-PCR kits (Results, Lines 207-212).
Overall, when detecting the diluted samples higher than the claimed LODs, the 20-in-1 pooling tests could substantially achieve the same detection performance as the single sampling tests. When it came to Delta (22/27 vs 15/27, P = 0.040) and Omicron (22/27 vs 14/27, P = 0.021) variants lower than the claimed LODs, the impaired analytical sensitivity was found in the 20-in-1 pooling strategy using Kit N03 (Results, Lines 215-219).
Discussion: Based on the results above, we discussed the results in the Discussion section: When using five rRT-PCR kits to detect the same strain, the range of lowest inactivated virus concentrations to achieve 100% detection by 20-in-1 pooling tests was substantially the same as single sampling tests. Overall, when detecting the diluted samples higher than the claimed LODs, 20-in-1 pooling tests showed comparable analytical sensitivity to the single sampling tests (Discussion, Lines 298-302). Therefore, the 20-in-1 pooling tests enable the mass nucleic acid tests with only 5% of the original testing workload, greatly improving daily testing efficiency, which is the first consideration in the screening of asymptomatic infected cases in low-risk regions (Discussion, Lines 304-307).
(2) Point-of care test Results: Unexpectedly, the POCT Kit P01 showed comparable analytical sensitivity to rRT-PCR kits with the lowest concentration of 1.42×10 3 copies/mL for all three strains ( (

3) Rapid antigen tests
Results: On testing of inactivated cell culture supernatants, the lowest virus concentrations to achieve a 100% detection rate ranged between 2.84×10 5 -7.14×10 6 , 8.68×10 4 -7.14×10 6 , and 1.12×10 5 -3.57×10 6 copies/mL for the WT, Delta and Omicron strains, respectively, corresponding to 3.12×10 6 -5.00×10 7 , 7.81×10 5 -5.00×10 7 , and 7.81×10 5 -2.50×10 7 copies/mL of the 50 μL original sample ( Different from the rRT-PCR kits, great variations in analytical sensitivity were observed among rapid antigen kits. The lowest concentrations that achieved a 100% rate of detection success ranged from 10 4 to 10 6 copies/mL for inactivated cell culture supernatants and from 1 to 150 ng/mL for recombinant N proteins. The best analytical performance was achieved by Kit A05 in detecting inactivated viruses, while detecting recombinant N proteins, Kit A01 performed the best. The assay manufactured by Kit A19 was considerably less sensitive than the other assays both in detecting inactivated viruses and recombinant N proteins (Results, Lines 236-243).
Discussion: Based on the results above, we concluded in the Discussion section: Our study revealed that the detection performance of different rapid antigen kits varied greatly, and the lowest concentrations that achieved a 100% rate of detection success ranged from 10 4 to 10 6 copies/mL for inactivated cell culture supernatants and from 1 to 150 ng/mL for recombinant N proteins, which was essentially consistent with previously published articles. The antibody labeling method of rapid antigen tests would affect their analytical performance, and the fluorescence microsphere was observed to enhance the sensitivity of analytical signal by 10 to 100 folds compared to latex microsphere and colloidal gold (40). Besides, the lowest concentrations of the 50 μL original sample at 100% detection of rapid antigen tests (10 5 -10 7 copies/mL) were about 100 times as high as those of the single sampling test (10 3 -10 5 copies/mL) In the end, we summarized the sensitivity difference among the four detection strategies obtained in this evaluation and provided helpful insights into their scientific deployment in the last paragraph of the article (Discussion, Lines 382-389): Generally, the analytical sensitivity of nucleic acid amplification tests was superior to that of rapid antigen tests, with single sampling strategy showing the highest. In a specific scenario, the optimal strategy should be adopted in consideration of the testing purpose, resource availability, cost performance and result rapidity on the premise of test accuracy, thereby improving overall detection efficiency, facilitating the discovery of early community transmission, and enabling timely and long-term infection control measures under the condition of limited detection capacity and overburdened laboratory infrastructure.
Comment 2: figure 2 besides being hard to follow, the way it is presented makes it difficult to have a comprehensive appreciation of the results.
Answer 2: Thank you for your valuable suggestion.
To reduce unnecessary confusion, we decided to use tables mainly to present the results and delete Figure 2. Table 3 and Table 4 showed the lowest concentrations of samples to achieve 100% detection rates. Besides, to provide more detailed information in the study, we have added Supplementary Material File 3 attached with the raw data of test。 Besides, to provide more detailed information in the study, we Comment 3: Authors should say how the limit of detection for each test was calculated.
Answer 3: Thank you for your valuable suggestion.
For the single sampling tests and 20-in-1 pooling tests, 50 μL cell culture supernatants were added to 3 mL and 12 mL sample preservation solution, resulting in 61-fold and 241-fold dilutions of the initial sample concentration, respectively. The "concentration equivalent to the 50 μL sample" in Table 3 was the lowest concentration to achieve 100% detection rates of the 50 μL original sample before being added to the sample preservation solution. The "lowest concentration at 100% detection rates" was the lowest concentration to achieve 100% detection rates of the diluted sample after being added to the sample preservation solution, and was 1/61 (single sampling tests) or 1/241 (20-in-1 pooling tests) of the 50 μL original concentration.
For the point-of-care test, 50 μL cell culture supernatants were added to 500 μL sample extraction solution, resulting in an 11-fold dilution of the initial sample concentration. Similar to the rRT-PCR kits, the "lowest concentration at 100% detection rates" was 1/11 of the "concentration equivalent to the 50 μL sample" in Table 3.
For the rapid antigen tests, 50 μL cell culture supernatants or recombinant N proteins were added to different volumes of sample extraction solution for different antigen tests. Taking BGI as an example, 50 μL cell culture supernatants or recombinant N proteins were added to 400 μL sample extraction solution resulting in a 9-fold dilution of the initial sample concentration. Therefore, the "lowest concentration at 100% detection rates" was 1/9 of the "concentration equivalent to the 50 μL sample" in Table 3 and Table 4.
In order to describe the pre-dilution ratio and the calculation of the LOD for each kit more clearly, we have added " Table 2 Calculation of pre-dilution ratio of different detection methods." Final volume Pre-dilution ratio